Flavivirus neutralizing antibodies and methods of use thereof

ABSTRACT

The present invention provides antibodies that neutralize flavivirus and methods of use thereof. These antibodies are derived from mAbl 1 which recognizes West Nile virus E protein and is cross-reactive with members of the flavivirus family, including Denge virus. The antibodies of the present invention prevent antibody-dependent enhancement of a viral infection by having a modified Fc region that does not bind to the Fcy receptor. The invented antibody is used to treat flaviviral infections and symptoms thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 15/818,491, filed on Nov. 20, 2017, now issued as U.S. Pat. No.10,400,032, which is a divisional application of U.S. patent applicationSer. No. 14/777,324, filed on Sep. 15, 2015, now issued as U.S. Pat. No.9,822,166, which is a national stage application filed under 35 U.S.C. §371 of International Application No. PCT/US2014/028310, filed on Mar.14, 2014, which claims benefit of, and priority to, U.S. provisionalpatent application No. 61/792,336 filed on Mar. 15, 2013; the contentsof each which are hereby incorporated by reference in their entireties.

GOVERNMENT INTEREST

This invention was made with government support under grant numberAI070343 awarded by The National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The contents of the file named “DFCI-073_N01US 322270-2532_ST25.txt”,which was created on Sep. 15, 2015 and is 28 KB in size, are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally to flavivirus neutralizing antibodiesas well as to methods for use thereof.

BACKGROUND OF THE INVENTION

Flaviviruses, such as West Nile virus and Dengue virus, present asignificant threat to global health. West Nile virus causes a febrileillness that can lead to fatal meningitis or encephalitis acrossmultiple species. West Nile virus can be carried by both birds andmosquitos, which has allowed its spread at an alarming pace worldwide.Similarly, four serotypes of Dengue virus can be transmitted throughmosquito bites, and causes tens of millions of human cases of dengueannually, including 500,000 hospitalizations and 20,000 deaths, with aneconomic burden rivalling that of malaria.

Vaccines and antibody therapeutics are currently in development toprevent and treat flavivirus infection. However, evidence from denguevirus infections indicate that vaccination strategies for flavivirusesmay not be as straightforward as other viruses. A first infection withone Dengue virus serotype induces protective immunity to the homologousserotype. However, there is no cross-protection against infection by adifferent serotype. Instead, pre-existing immunity is associated withincreased risk of infection and illness due to antibody-dependentenhancement (ADE) of infection. In ADE, antibodies raised by priorflavivirus infection or passively transferred from mother to child causean increased rate of infection and pathogenicity. Thus, conventionalmethods of antibody-based therapeutics or vaccines against flavivirusmay exponentially increase incidence of flavivirus infection andillness.

Accordingly, there is an urgent need for therapeutics and methods forpreventing flavivirus infection, and diseases and disorders relatedthereto, without increasing the risk of antibody-dependent enhancementof infection.

SUMMARY OF THE INVENTION

The invention is based upon the discovery of monoclonal antibodies whichbind and neutralize flavivirus, and do not contribute to theantibody-dependent enhancement of flavivirus infection. The monoclonalantibody is fully human. The antibodies recognize the West Nile virusenvelope protein E (WNE), and have broad cross-reactivity across othermembers of the flavivirus family. Importantly, the antibodies containmutations in the Fc region that prevent binding to the Fcγ receptor. Theantibodies are referred to herein as huFV antibodies.

The invention provides an isolated humanized monoclonal antibody havinga heavy chain with three CDRs, wherein the CDR1 includes amino acidsequence GYSTH (SEQ ID NO:21), wherein the CDR2 includes amino acidsequence WDNPSSGDTTYAENFRG (SEQ ID NO:22), and wherein the CDR3 includesamino acid sequence GGDDYSFDH (SEQ ID NO:23) respectively; a light chainwith three CDRs, wherein the CDR1 includes amino acid sequenceRGDSLRSYYAS (SEQ ID NO:24), wherein the CDR2 includes amino acidsequence GENNRPS (SEQ ID NO:25), and wherein the CDR3 includes aminoacid sequence NSRDSSDHLLL (SEQ ID NO:26) respectively. The antibody hasa modified Fc region such that the Fc region does not bind to the Fcγreceptor, and binds to a flavivirus. Exemplary Fc regions are disclosedherein.

In one aspect, the invention provides an isolated humanized monoclonalantibody having a V_(H) amino acid sequence having SEQ ID NO: 1, a V_(L)amino acid sequence having SEQ ID NO: 3. In another aspect, theinvention provides an isolated humanized monoclonal antibody comprisinga V_(H) nucleotide sequence having SEQ ID NO: 2, a V_(L) nucleotidesequence having SEQ ID NO: 4. The antibody further comprises a modifiedFc region such that the Fc region does not bind to the Fcγ receptor, andbinds to a flavivirus.

The present invention provides an isolated humanized monoclonal antibodythat neutralizes a flavivirus.

The present invention provides antibodies with a modified Fc region suchthat the Fc region does not bind to the Fcγ receptor. The modified Fcregion contains mutations at amino acid positions 234 and 235. In oneaspect, the mutations are L234A and L235A. In one embodiment, themodified Fc region comprises a CH2 region wherein the amino acids atpositions 4 and 5 of the CH2 region are mutated. For example, theleucine amino acids at positions 4 and 5 are mutated to a differentamino acid, preferably, an alanine. In other embodiments, the modifiedFc region comprises an Fc region where the amino acids at positions 108and 109 are mutated. For example, the leucines at positions 108 and 109of the Fc region are mutated to a different amino acid, preferably, analanine. In another embodiment, the modified Fc region comprises theamino acid sequence of SEQ ID NO: 7. The modified Fc region binds to theneonatal Fc receptor (FcRn).

In one aspect, the antibody does not contribute to an antibody-dependentenhancement of a flavivirus infection.

In one aspect, the antibody is linked to a therapeutic agent. Thetherapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule,or a cytokine. For example, the cytokine is TGF-beta.

The present invention further provides a cell producing a huFV antibody.The cell may be a mammalian cell (i.e., a mouse, rabbit, goat, orsheep), or a plant cell (i.e. tobacco plant).

The present invention provides a method for preventingantibody-dependent enhancement of a flavivirus infection byadministering a huFv antibody to a subject. In one aspect, the antibodyis administered after a first infection by a flavivirus.

Additionally, the present invention provides a method of increasingvaccine efficiency by administering to a subject a huFV antibody and avaccine. In one aspect, the huFV antibody and the vaccine areadministered sequentially or concurrently. In one aspect, the vaccine isa viral vaccine.

The present invention further provides a method for treating oralleviating a symptom of a flavivirus infection by administering to asubject in need thereof a composition containing a huFV antibody. Inanother aspect, the present invention features a method for delaying theonset or progression of one or more symptoms of a flavivirus infection.Symptoms of flavivirus infection include, but are not limited to, weightloss, paralysis, fever, headaches, nausea, vomiting, skin rash, and bodyaches. In one aspect, an anti-viral agent is also administered to thesubject. In one aspect, the huFV antibody and the anti-viral agent areadministered sequentially or concurrently. The anti-viral agent is anantibody, an antibody linked to a therapeutic agent, or a smallmolecule.

The flavivirus is West Nile virus, Dengue virus (serotypes 1-4), St.Louis encephalitis virus, yellow fever virus, Japanese encephalitisvirus, and Murray Valley encephalitis virus.

The invention further provides a nucleic acid sequence containing anucleic acid sequence of SEQ ID NO: 2, 4 and 8.

The invention further provides a nucleic acid sequence encoding apolypeptide of SEQ ID NO: 1 and 3. The invention further provides apolypeptide containing the amino acid sequence of SEQ ID NO: 1, 3 and 7.

The invention further provides a vector containing the nucleic acidsequence containing SEQ ID NO: 2, 4 and 8, or encoding a polypeptide ofSEQ ID NO: 1, 3 and 7.

Additionally, the invention provides a cell containing a vectorcontaining the nucleic acid sequence containing SEQ ID NO: 2, 4 and 8,or encoding a polypeptide of SEQ ID NO: 1, 3 and 7.

Other features and advantages of the invention will be apparent from andare encompassed by the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of an expression vector containing wild-typemAb-11.

FIG. 2 shows a schematic of an expression vector containing engineeredmAb-11-LALA.

FIG. 3 is a series of three graphs showing the mAb11(LALA) protectionagainst lethal Dengue 2 virus infection. (A) Kaplan Meier survival curvecompares the survival of mice administered with mAb11 with wild-type(wt) or mutant (mut) Fc regions. (B) Percent of original weight wasmonitored in mice administered with mAb11 with wild-type (wt) or mutant(mut) Fc regions. (C) Clinical scores were determined based onappearance, mobility and general attitude of the mice administered withmAb11 with wild-type (wt) or mutant (mut) Fc regions.

FIG. 4 is a series of three graphs comparing the survival rate of miceadministered mammalian mAb11 with LALA mutation (mutAb) derived frommammalian cells or plant cells, and then challenged with lethal Dengue 2virus infection. (A) Mammalian-derived mutant mAb11 antibody compared toplant-derived mutant mAb11 antibody. (B) Mammalian-derived mutant mAb11antibody compared to control antibody. (C) Plant-derived mutant mAb11antibody compared to control antibody.

FIG. 5 is a series of three graphs comparing the percent weight of miceadministered mammalian mAb11 with LALA mutation (mutAb) derived frommammalian cells or plant cells, and then challenged with lethal Dengue 2virus infection. (A) Mammalian-derived mutant mAb11 antibody compared toplant-derived mutant mAb11 antibody. (B) Mammalian-derived mutant mAb11antibody compared to control antibody. (C) Plant-derived mutant mAb11antibody compared to control antibody.

FIG. 6 is a series of three graphs comparing the clinical score of miceadministered mammalian mAb11 with LALA mutation (mutAb) derived frommammalian cells or plant cells, and then challenged with lethal Dengue 2virus infection. (A) Mammalian-derived mutant mAb11 antibody compared toplant-derived mutant mAb11 antibody. (B) Mammalian-derived mutant mAb11antibody compared to control antibody. (C) Plant-derived mutant mAb11antibody compared to control antibody.

DETAILED DESCRIPTION

The present invention provides antibodies that neutralize infection bymembers of the flavivirus family without contributing toantibody-dependent enhancement of virus infection. Antibodies that bindand neutralize West Nile virus are described in InternationalPublication WO 2005/123774, the contents of which are incorporated byreference in its entirety. The antibodies of the present invention wereproduced by modifying an antibody against West Nile virus, mAb11, suchthat the Fc region of the antibody does not bind to the Fc-gammareceptor. Thus, the modified antibody does not contribute toantibody-dependent enhancement of infection. The antibodies and methodsdisclosed herein relate to this antibody, and methods for treating orpreventing an infection by a flavivirus, and related diseases anddisorders. The antibody of the present invention, mAB11-LALA hasdemonstrated increased capability of preventing and treating flavivirusinfection compared to the wild-type mAb11, as described herein anddemonstrated in the examples.

MAb11 was identified using a phage display screening and binds to theWest Nile virus envelope protein E, within the DI and DII domains andspecifically at the fusion loop peptide (Gould et al., Journal ofVirology, 2005, 79(23):14606-14613; Sultana et al., Journal ofImmunology, 2009, 183: 650-660, the contents of which are incorporatedherein in their entirety). The engineered mAb11 antibody with mutationsat amino acids positions 234 and 235 in the Fc region is referred toherein as “mAb11-LALA”.

The antibodies of the present invention have broad cross-reactivity tothe members of the Flavivirus family. For example, the antibodydemonstrates cross-reactivity and neutralization of several differentflaviviruses, including, but not limited to West Nile virus, Denguevirus (serotypes 1, 2, 3 and 4), St. Louis encephalitis virus, yellowfever virus, Japanese encephalitis virus, and Murray Valley encephalitisvirus.

Neutralizing antibodies have been and are being currently developed forthe treatment and prevention of viral infections, specificallyinfections by members of the Flavivirus genus. Initial studies havedemonstrated that such antibodies show increased neutralization andprotection from infection by flavivirus family members (i.e., West NileVirus or one of the four Dengue virus serotypes). However, subsequentvirus challenge studies, in which experimental subjects were treatedwith neutralizing antibodies and then challenged with doses offlavivirus (i.e., Dengue virus), did not show a decrease in viremia. Insome cases, treatment with such antibodies resulted in enhancement ofinfection compared to controls, which is believed to be mediated thougha mechanism called antibody-dependent enhancement (ADE). These resultsdemonstrate the importance of developing therapeutics and methods thatprevent antibody-dependent enhancement of flavivirus infection.

Antibody-dependent enhancement of infection can be accomplished by thebinding of the Fc region of the antibody to an Fcγ receptor (FcγR) on ahost cell. Infectious viral particles bound to these antibodies aretherefore more efficiently brought to host cells by Fc region-Fcreceptor binding. This increases the infection and replication rate ofthe virus, thereby enhancing the infectivity and pathogenicity of thevirus.

In contrast to standard anti-viral antibodies, the antibodies of thepresent invention have reduced binding to the Fcγ receptors (FcγR) or donot bind to the FcγR. Fcγ receptors include, for example, FcγRI,FcγRIIIa, FcγRIIIb, and FcγRIIIc. In one embodiment, the antibodies ofthe invention contain one or more mutations in the Fc region. Themutation(s) may be any mutation that reduces or abrogates binding of theantibody to a FcγR. Mutations can be substitutions, additions, ordeletions of amino acids in the Fc region. Although the antibodies ofthe present invention have mutated Fc regions, the antibodies stillconfer potent flavivirus neutralization.

The Fc region of an antibody comprises two domains, CH2 and CH3. Thesedomains, or specific amino acids within these domains known in the art,mediate the interaction with FcγR. Antibodies of the present inventioncontain any mutation (i.e., substitution, addition, or deletion of oneor more than one amino acid) in the CH2 or CH3 domain, or both, thatreduces or abrogates the binding of the antibody to an FcγR. Forexample, antibodies of the present invention contain a mutation orsubstitution of at least one amino acid at positions 233, 234, 235, 236,237, 250, 314, or 428 of the wild-type Fc region. Preferably, the aminoacid substitution is to an alanine.

In one embodiment, the Fc region of an antibody of the inventioncomprises a substitution at positions 234 or 235 of the heavy chain ofthe antibody, or both. In general, the amino acid at positions 234 and235 of the wild-type Fc region is a leucine (“L”). In one embodiment,the antibodies of the invention comprise an amino acid at position 234,235, or both, that is not a leucine. In another embodiment, theantibodies of the invention comprise an alanine (“A”) at position 234,235 or both. An antibody comprising the mutations at positions 234 and235 of the Fc region where the leucines are mutated to alanines isreferred to herein as a “LALA” variant.

In a preferred embodiment, the antibodies of the present invention arefull length, or intact, antibodies, wherein the antibodies contain anantigen-binding region (i.e., Fab region or Fab fragment) and an Fcregion (modified or mutated, as described herein). Previously developedantibodies in the art that were designed to circumvent ADE often lackthe Fc region to prevent binding to FcγR. Antibodies of the presentinvention provide superior properties by retaining the Fc region. Onesuch property is the ability to bind to the neonatal receptor (FcRn)expressed on endothelial cells, which plays a critical role in thehomeostasis of circulating IgG levels. Binding of circulating antibodiesto the FcRn induces internalization through pinocytosis, in which theantibodies are recycled to the cell surface, and released at the basicpH of blood. This mechanism protects the antibodies of the presentinvention from degradation and increases the half-life compared to otherunmodified antibodies or antibody fragments lacking the Fc region.Increased persistence of the antibodies of the present invention in theserum provides increased efficacy by allowing higher circulating levels,less frequent administration, and reduced doses. Another property of theantibodies of the present invention may include the ability to bind tocomplement factors. Binding of complement factors, such as C1q, to theFc region of the antibody triggers a signaling cascade to activatecomplement-dependent cytotoxicity (CDC).

It is known in the art that the binding sites on the Fc region of Fcγreceptors is distinct from the binding site of the neonatal Fc receptor(FcRn). Therefore, the antibodies of the present invention have Fcregions modified such that they have reduced binding or cannot bind tothe Fcγ receptors, however are still competent for binding to the FcRnreceptor. Antibodies of the invention can be modified by introducingrandom amino acid mutations into particular region of the CH2 or CH3domain of the heavy chain in order to alter their binding affinity forFcγR and/or FcRn and/or their serum half-life in comparison to theunmodified antibodies. Examples of such modifications include, but arenot limited to, substitutions of at least one amino acid from the heavychain region selected from the group consisting of amino acid residues234, 235, 236, 237, 250, 314, and 428. Accordingly, the antibodies ofthe present invention have greater half-life than unmodified antibodies,which confers increased efficacy in the prevention and treatment offlavivirus infections and subsequent disease.

In one aspect, the antibodies of the present invention have Fc regionsmodified such that have reduced binding or cannot bind to the Fcγreceptors, however are still competent for binding to complementfactors, such as C1q.

One of ordinarily skill in the art could readily prepare the modifiedantibodies of the present invention. Recombinant DNA techniques forintroducing mutations or substitutions in the Fc region of an antibodyare known in the art. Characterization of the Fc region for theirability to bind or not bind to Fc receptors (Fcγr or FcRn) can bereadily performed by the ordinarily skilled artisan, for example byimmunoprecipitation, immunoassay, affinity chromatography, or arraytechniques.

The humanized antibodies described herein may be produced in mammalianexpression systems, such as hybridomas. The humanized antibodiesdescribed herein may also be produced by non-mammalian expressionsystems, for example, by transgenic plants. For example, the antibodiesdescribed herein are produced in transformed tobacco plants (N.benthamiana and N. tabaccum).

The various nucleic acid and amino acid sequences of mAb11 of thepresent invention is provided below:

Heavy Chain Variable (V_(H)) Amino Acid Sequence: (SEQ ID NO: 1)TRVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFSGYSTHWLRQVPGQGLEWIGWDNPSSGDTTYAENFRGRVTLTRDTSITTDYLEVRGLRSDDTAVYYCARGGDDYSFDHWGQGTLVTVSSHeavy Chain Variable (V_(H)) Nucleic Acid Sequence: (SEQ ID NO: 2)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaagtctectgcaaggcttctggatacaccttcageggctactctacacactggctgcgacaggtecctggacagggacttgagtggattggatgggacaaccctagtagtggtgacacgacctatgeagagaatfficggggcagggtcaccetgaccagggacacgtccatcaccacagattacttggaagtgaggggtetaagatctgacgacacggccgtetattattglgccagaggeggagatgactacagctttgaccattggggtcagggcacc ctggtcaccgtctcctcaLight Chain Variable (V_(H)) Amino Acid Sequence: (SEQ ID NO: 3)SSELTQDPAVSVALGQTVRITCRGDSLRSYYASWYQQKPGQAPVLVIYGENNRPSGIPDRFSGSSSGDTASLTITGAQAEDEADYYCNSRDSSDHL LLFGQGTKLLight Chain Variable (V_(H)) Nucleic Acid Sequence: (SEQ ID NO: 4)Tcttctgagctgactcaggacccagctgtgtctgtggccttgggacagacagtcaggatcacatgccgaggagacagcctcagaagttattatgcaagctggtaccaacagaagccaggacaggcccctgtacttgtcatctatggtgaaaacaaccgaccctcagggatcccagaccgattctctggctccagctcaggagacacagcttccttgaccatcactggggctcaggcggaagatgaggctgactattactgtaactcccgggacagcagtgatcaccttctcctattcggtggagggaccaagttgaccgtcctaggt

The Fc region comprises three heavy constant domains, CH1, CH2 or CH3domains. A hinge region joins the CH1 and CH2 regions. Exemplary Fcregion sequences for wild-type and modified Fc regions with respect tothe invention are provided below.

The amino acid sequence of the Fc Region of wild-type mAb-11 is providedas follows:

(SEQ ID NO: 5) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The nucleic acid sequence of the Fc Region of wild-type mAb-11 isprovided as follows:

(SEQ ID NO: 6) CTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The amino acid sequence of the modified Fc region of mAb-11-LALA isprovided below. For example, the amino acids at positions 108 and 109are mutated. In the sequence provided below, the leucine amino acids atposition 108 and 109 are mutated to alanines (underlined).

(SEQ ID NO: 7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The nucleic acid sequence of the modified Fc region of mAb-11-LALA isprovided below. For example, the amino acids at positions 108 and 109encoded by the provided nucleic acid sequence are mutated from leucinesto alanines (underlined).

(SEQ ID NO: 8) CTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The amino acid sequence for the CH1 region is provided below:

(SEQ ID NO: 9) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK

The nucleic acid sequence for the CH1 region is provided below:

(SEQ ID NO: 10) CTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAA

The mAb11 antibody described herein may comprise a mutation specificallyin the CH2 region that reduces or inhibits binding to the Fcγ receptor.Preferably, the mutation does not affect binding to FcRn receptor.Preferably, the mAb11 antibody contains two mutations in the CH2 region,such that two adjacent lysines are mutated to alanines described below.For example, the mutations are located at amino acid positions 4 and 5of the CH2 region. Preferably, the mutations are to alanines.

The amino acid sequence for the CH2 region of the wild-type mAb11antibody is provided below:

(SEQ ID NO: 11) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAK

The nucleic acid sequence for the CH2 region of the wild-type mAb11antibody is provided below:

(SEQ ID NO: 12) GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA

The amino acid sequence for the CH2 region of the mutant mAb11 antibodyis provided below (the LALA mutation is underlined):

(SEQ ID NO: 13) APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAK

The nucleic acid sequence for the CH2 region of the mutant mAb11antibody is provided below (the LALA mutation is underlined):

(SEQ ID NO: 14) GCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA

The amino acid sequence for the CH3 region of the mAb11 antibody isprovided below:

(SEQ ID NO: 15) GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

The nucleic acid sequence for the CH3 region of the mAb11 antibody isprovided below:

(SEQ ID NO: 16) GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The amino acid sequence for the hinge region is provided below:

(SEQ ID NO: 17) AEPKSCDKTHTCPPCP

The nucleic acid sequence for the hinge region is provided below:

(SEQ ID NO: 18) GCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCA

The amino acid sequence of the heavy chain (including both variable andconstant regions) of wild-type mAb-11 is provided below:

(SEQ ID NO: 19) TRVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFSGYSTHWLRQVPGQGLEWIGWDNPSSGDTTYAENFRGRVTLTRDTSITTDYLEVRGLRSDDTAVYYCARGGDDYSFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK

The amino acid sequence of the heavy chain (including both variable andconstant regions) of mutant mAb-11 is provided below (LALA mutation isunderlined):

(SEQ ID NO: 20) TRVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFSGYSTHWLRQVPGQGLEWIGWDNPSSGDTTYAENFRGRVTLTRDTSITTDYLEVRGLRSDDTAVYYCARGGDDYSFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKAntibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” is meant that the antibody reacts with one or moreantigenic determinants of the desired antigen and does not react withother polypeptides. Antibodies include, but are not limited to,polyclonal, monoclonal, and chimeric antibodies

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain. The term“antigen-binding site,” or “binding portion” refers to the part of theimmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, a scFv, or a T-cellreceptor. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. For example, antibodies maybe raised against N-terminal or C-terminal peptides of a polypeptide.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to a flavivirus epitope when the equilibriumbinding constant (K_(d)) is ≤1 μM, preferably ≤100 nM, more preferably≤10 nM, and most preferably ≤100 μM to about 1 μM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

An flavivirus protein (i.e., an envelope protein or West Nile envelopeprotein E) of the invention, or a derivative, fragment, analog, homologor ortholog thereof, may be utilized as an immunogen in the generationof antibodies that immunospecifically bind these protein components.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the inventionby ascertaining whether the former prevents the latter from binding toflavivirus. If the human monoclonal antibody being tested competes withthe human monoclonal antibody of the invention, as shown by a decreasein binding by the human monoclonal antibody of the invention, then it islikely that the two monoclonal antibodies bind to the same, or to aclosely related, epitope.

Another way to determine whether a human monoclonal antibody has thespecificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with theflavivirus envelope proteins, such as West Nile virus protein E, withwhich it is normally reactive, and then add the human monoclonalantibody being tested to determine if the human monoclonal antibodybeing tested is inhibited in its ability to bind flavivirus envelopeproteins, such as West Nile virus E. If the human monoclonal antibodybeing tested is inhibited then, in all likelihood, it has the same, orfunctionally equivalent, epitopic specificity as the monoclonal antibodyof the invention. Screening of human monoclonal antibodies of theinvention, can be also carried out by utilizing WNE and determiningwhether the test monoclonal antibody is able to neutralize members ofthe flavivirus family.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof. (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference).

Antibodies can be purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The term “monoclonal antibody” or “MAb” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanizedantibodies”, “human antibodies”, or “fully human antibodies” herein.Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

In addition, humanized antibodies can be produced in transgenic plants,as an an inexpensive production alternative to existing mammaliansystems. For example, the transgenic plant may be a tobacco plant, i.e.,Nicotiania benthamiana, and Nicotiana tabaccum. The antibodies arepurified from the plant leaves. Stable transformation of the plants canbe achieved through the use of Agrobacterium tumefaciens or particlebombardment. For example, nucleic acid expression vectors containing atleast the heavy and light chain sequences are expressed in bacterialcultures, i.e., A. tumefaciens strain BLA4404, via transformation.Infiltration of the plants can be accomplished via injection. Solubleleaf extracts can be prepared by grinding leaf tissue in a mortar and bycentrifugation. Isolation and purification of the antibodies can bereadily be performed by many of the methods known to the skilled artisanin the art. Other methods for antibody production in plants aredescribed in, for example, Fischer et al., Vaccine, 2003, 21:820-5; andKo et al, Current Topics in Microbiology and Immunology, Vol. 332, 2009,pp. 55-78. As such, the present invention further provides any cell orplant comprising a vector that encodes the antibody of the presentinvention, or produces the antibody of the present invention.

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen and a correlative method forselecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence offlavivirus in a sample. The antibody can also be used to try to bind toand disrupt flavivirus envelope protein activity.

In a preferred embodiment, the antibodies of the present invention arefull-length antibodies, containing an Fc region similar to wild-type Fcregions that bind to Fc receptors.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in neutralizing or preventing viral infection. For example,cysteine residue(s) can be introduced into the Fc region, therebyallowing interchain disulfide bond formation in this region. Thehomodimeric antibody thus generated can have improved internalizationcapability and/or increased complement-mediated cell killing andantibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J.Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922(1992)). Alternatively, an antibody can be engineered that has dual Fcregions and can thereby have enhanced complement lysis and ADCCcapabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230(1989)). In a preferred embodiment, the antibody of the presentinvention has modifications of the Fc region, such that the Fc regiondoes not bind to the Fc receptors. Preferably, the Fc receptor is Fcγreceptor. Particularly preferred are antibodies with modification of theFc region such that the Fc region does not bind to Fcγ, but still bindsto neonatal Fc receptor.

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987)). Preferred linkers aredescribed in the literature. (See, for example, Ramakrishnan, S. et al.,Cancer Res. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against Flaviviruses

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against a flavivirus envelope protein such as WNE(or a fragment thereof) may be used in methods known within the artrelating to the localization and/or quantitation of a flavivirusenvelope protein such as WNE (e.g., for use in measuring levels of theflavivirus protein within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific to an flavivirus envelope proteinsuch as WNE, or derivative, fragment, analog or homolog thereof, thatcontain the antibody derived antigen binding domain, are utilized aspharmacologically active compounds (referred to hereinafter as“Therapeutics”).

An antibody specific for a flavivirus envelope protein such as WNE ofthe invention can be used to isolate a flavivirus polypeptide bystandard techniques, such as immunoaffinity, chromatography orimmunoprecipitation. Antibodies directed against an flavivirus protein(or a fragment thereof) can be used diagnostically to monitor proteinlevels in tissue as part of a clinical testing procedure, e.g., to, forexample, determine the efficacy of a given treatment regimen. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, (3-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may used as therapeutic agents. Such agentswill generally be employed to treat or prevent a flavivirus-relateddisease or pathology (e.g., dengue fever) in a subject. An antibodypreparation, preferably one having high specificity and high affinityfor its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withthe internalization of the virus into a cell. In this case, the antibodybinds to the target and prevents binding to an Fc receptor-expressingcell, thereby blocking fusion the virus to the cell membrane inhibitinginternalization of the virus in antibody-dependent enhancement ofinfection.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding a flavivirus protein or a fragmentthereof of the invention, as well as other molecules identified by thescreening assays disclosed herein, can be administered for the treatmentof a flavivirus-related disorders in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhome, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

In the embodiments of the present invention, antibody fragments are notpreferred, specifically antibody fragments lacking an Fc region. Peptidemolecules can be designed that retain the ability to bind the targetprotein sequence. Such peptides can be synthesized chemically and/orproduced by recombinant DNA technology. (See, e.g., Marasco et al.,Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation canalso contain more than one active compound as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. Alternatively, or inaddition, the composition can comprise an agent that enhances itsfunction, such as, for example, a cytotoxic agent, cytokine,chemotherapeutic agent, or growth-inhibitory agent. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of a flavivirus (or a protein or a proteinfragment thereof) in a sample. Preferably, the antibody contains adetectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody is preferred. The term “labeled”, withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (i.e., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently-labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently-labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. Included within the usage of the term “biologicalsample”, therefore, is blood and a fraction or component of bloodincluding blood serum, blood plasma, or lymph. That is, the detectionmethod of the invention can be used to detect an analyte mRNA, protein,or genomic DNA in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of an analyte mRNA includeNorthern hybridizations and in situ hybridizations. In vitro techniquesfor detection of an analyte protein include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. In vitro techniques for detection of an analytegenomic DNA include Southern hybridizations. Procedures for conductingimmunoassays are described, for example in “ELISA: Theory and Practice:Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) HumanPress, Totowa, N.J., 1995; “Immunoassay”, E. Diamandis and T.Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and“Practice and Theory of Enzyme Immunoassays”, P. Tijssen, ElsevierScience Publishers, Amsterdam, 1985. Furthermore, in vivo techniques fordetection of an analyte protein include introducing into a subject alabeled anti-analyte protein antibody. For example, the antibody can belabeled with a radioactive marker whose presence and location in asubject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or agents of the invention (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the antibody oragent and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with the fusion of anflavivirus to the cell membrane. Also provided are methods ofidentifying compounds useful to treat flavivirus infection. Theinvention also encompasses compounds identified using the screeningassays described herein.

For example, the invention provides assays for screening candidate ortest compounds which modulate the interaction between the flavivirus andthe cell membrane. The test compounds of the invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the “one-bead one-compound” librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds. (See,e.g., Lam, 1997. Anticancer Drug Design 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates the interactionbetween a flavivirus and the cell membrane. For example, the antibodymay be monoclonal antibody mAb11, mAb11-LALA, or any variant thereof,and the antigen may be located on an envelope protein of a flavivirus(i.e., West Nile virus protein E).

In another embodiment, at least one flavivirus envelope protein isprovided, which is exposed to at least one neutralizing monoclonalantibody. Formation of an antibody-antigen complex is detected, and oneor more candidate compounds are introduced to the complex. If theantibody-antigen complex is disrupted following introduction of the oneor more candidate compounds, the candidate compounds is useful to treata flavivirus-related disease or disorder, e.g. Dengue fever. Forexample, the at least one flavivirus protein may be provided as aflavivirus molecule.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a flavivirus neutralizingantibody, such as monoclonal antibody Ab-11 or any variant thereofwherein the Fc region is modified such that it has reduced binding ordoes not bind to the Fc-gamma receptor. Additionally, the antigen may bea flavivirus envelope protein, or a portion thereof.

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. The cell-free assays of the invention areamenable to use of both the soluble form or the membrane-bound form ofthe proteins and fragments thereof. In the case of cell-free assayscomprising the membrane-bound forms of the proteins, it may be desirableto utilize a solubilizing agent such that the membrane-bound form of theproteins are maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody or the antigen (e.g. the can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated antibody or antigenmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques well-known within the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,other antibodies reactive with the antibody or antigen of interest, butwhich do not interfere with the formation of the antibody-antigencomplex of interest, can be derivatized to the wells of the plate, andunbound antibody or antigen trapped in the wells by antibodyconjugation. Methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using such other antibodies reactive withthe antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic Assays

Antibodies of the present invention can be detected by appropriateassays, e.g., conventional types of immunoassays. For example, an assaycan be performed in which a flavivirus envelope protein (e.g., West Nilevirus protein E) or fragment thereof is affixed to a solid phase.Incubation is maintained for a sufficient period of time to allow theantibody in the sample to bind to the immobilized polypeptide on thesolid phase. After this first incubation, the solid phase is separatedfrom the sample. The solid phase is washed to remove unbound materialsand interfering substances such as non-specific proteins which may alsobe present in the sample. The solid phase containing the antibody ofinterest bound to the immobilized polypeptide is subsequently incubatedwith a second, labeled antibody or antibody bound to a coupling agentsuch as biotin or avidin. This second antibody may be anotheranti-flavivirus antibody or another antibody. Labels for antibodies arewell-known in the art and include radionuclides, enzymes (e.g. maleatedehydrogenase, horseradish peroxidase, glucose oxidase, catalase),fluors (fluorescein isothiocyanate, rhodamine, phycocyanin,fluorescarmine), biotin, and the like. The labeled antibodies areincubated with the solid and the label bound to the solid phase ismeasured. These and other immunoassays can be easily performed by thoseof ordinary skill in the art.

An exemplary method for detecting the presence or absence of aflavivirus (in a biological sample involves obtaining a biologicalsample from a test subject and contacting the biological sample with alabeled monoclonal antibody according to the invention such that thepresence of the flavivirus is detected in the biological sample.

As used herein, the term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.That is, the detection method of the invention can be used to detect anflavivirus in a biological sample in vitro as well as in vivo. Forexample, in vitro techniques for detection of a flavivirus includeenzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. Furthermore, in vivotechniques for detection of a flavivirus include introducing into asubject a labeled anti-flavivirus antibody. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. One preferred biological sample is a peripheral bloodleukocyte sample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence of aflavivirus in a biological sample. For example, the kit can comprise: alabeled compound or agent capable of detecting a flavivirus (e.g., ananti-flavivirus monoclonal antibody) in a biological sample; means fordetermining the amount of a flavivirus in the sample; and means forcomparing the amount of a flavivirus in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect a flavivirusin a sample.

Passive Immunization

Passive immunization has proven to be an effective and safe strategy forthe prevention and treatment of viral diseases. (See Keller et al.,Clin. Microbiol. Rev. 13:602-14 (2000); Casadevall, Nat. Biotechnol.20:114 (2002); Shibata et al., Nat. Med. 5:204-10 (1999); and Igarashiet al., Nat. Med. 5:211-16 (1999), each of which are incorporated hereinby reference)). Passive immunization using neutralizing human monoclonalantibodies could provide an immediate treatment strategy for emergencyprophylaxis and treatment of flavivirus infection and related diseasesand disorders while the alternative and more time-consuming developmentof vaccines and new drugs in underway.

Subunit vaccines potentially offer significant advantages overconventional immunogens. They avoid the safety hazards inherent inproduction, distribution, and delivery of conventional killed orattenuated whole-pathogen vaccines. Furthermore, they can be rationallydesigned to include only confirmed protective epitopes, thereby avoidingsuppressive T epitopes (see Steward et al., J. Virol. 69:7668 (1995)) orimmunodominant B epitopes that subvert the immune system by inducingfutile, non-protective responses (e.g. “decoy” epitopes). (See Garrityet al., J. Immunol. 159:279 (1997)).

Moreover, those skilled in the art will recognize that good correlationexists between the antibody neutralizing activity in vitro and theprotection in vivo for many different viruses, challenge routes, andanimal models. (See Burton, Natl. Rev. Immunol. 2:706-13 (2002); Parrenet al., Adv. Immunol. 77:195-262 (2001)). The data presented hereindemonstrate that the mAb-11 human monoclonal antibody and other mAbvariants can be further developed and tested in in vivo animal studiesto determine its clinical utility as a potent ADE inhibitor forprophylaxis and treatment of flavivirus infection and related diseasesand disorders.

Antigen-Ig Chimeras in Vaccination

It has been over a decade since the first antibodies were used asscaffolds for the efficient presentation of antigenic determinants tothe immune systems. (See Zanetti, Nature 355:476-77 (1992); Zaghouani etal., Proc. Natl. Acad. Sci. USA 92:631-35 (1995)). When a peptide isincluded as an integral part of an IgG molecule (e.g., the 11A or 256IgG1 monoclonal antibody described herein), the antigenicity andimmunogenicity of the peptide epitopes are greatly enhanced as comparedto the free peptide. Such enhancement is possibly due to the antigen-IgGchimeras longer half-life, better presentation and constrainedconformation, which mimic their native structures.

Moreover, an added advantage of using an antigen-Ig chimera is thateither the variable or the Fc region of the antigen-Ig chimera can beused for targeting professional antigen-presenting cells (APCs). Todate, recombinant Igs have been generated in which thecomplementarity-determining regions (CDRs) of the heavy chain variablegene (V_(H)) are replaced with various antigenic peptides recognized byB or T cells. Such antigen-Ig chimeras have been used to induce bothhumoral and cellular immune responses. (See Bona et al., Immunol. Today19:126-33 (1998)).

Chimeras with specific epitopes engrafted into the CDR3 loop have beenused to induce humoral responses to either HIV-1 gp120 V3-loop or thefirst extracellular domain (D1) of human CD4 receptor. (See Lanza etal., Proc. Natl. Acad. Sci. USA 90:11683-87 (1993); Zaghouani et al.,Proc. Natl. Acad. Sci. USA 92:631-35 (1995)). The immune sera were ableto prevent infection of CD4 SupT1 cells by HIV-1MN (anti-gp120 V3C) orinhibit syncytia formation (anti-CD4-D1). The CDR2 and CDR3 can bereplaced with peptide epitopes simultaneously, and the length of peptideinserted can be up to 19 amino acids long.

Alternatively, one group has developed a “troybody” strategy in whichpeptide antigens are presented in the loops of the Ig constant (C)region and the variable region of the chimera can be used to target IgDon the surface of B-cells or MHC class II molecules on professional APCsincluding B-cells, dendritic cells (DC) and macrophages. (See Lunde etal., Biochem. Soc. Trans. 30:500-6 (2002)).

An antigen-Ig chimera can also be made by directly fusing the antigenwith the Fc portion of an IgG molecule. You et al., Cancer Res.61:3704-11 (2001) were able to obtain all arms of specific immuneresponse, including very high levels of antibodies to hepatitis B viruscore antigen using this method.

DNA Vaccination

DNA vaccines are stable, can provide the antigen an opportunity to benaturally processed, and can induce a longer-lasting response. Althougha very attractive immunization strategy, DNA vaccines often have verylimited potency to induce immune responses. Poor uptake of injected DNAby professional APCs, such as dendritic cells (DCs), may be the maincause of such limitation. Combined with the antigen-Ig chimera vaccines,a promising new DNA vaccine strategy based on the enhancement of APCantigen presentation has been reported (see Casares, et al., ViralImmunol. 10:129-36 (1997); Gerloni et al., Nat. Biotech. 15:876-81(1997); Gerloni et al., DNA Cell Biol. 16:611-25 (1997); You et al.,Cancer Res. 61:3704-11 (2001)), which takes advantage of the presence ofFc receptors (FcγRs) on the surface of DCs.

It is possible to generate a DNA vaccine encoding an antigen (Ag)-Igchimera. Upon immunization, Ag-Ig fusion proteins will be expressed andsecreted by the cells taking up the DNA molecules. The secreted Ag-Igfusion proteins, while inducing B-cell responses, can be captured andinternalized by interaction of the Fc fragment with FcγRs on DC surface,which will promote efficient antigen presentation and greatly enhanceantigen-specific immune responses. Applying the same principle, DNAencoding antigen-Ig chimeras carrying a functional anti-MHC II specificscFv region gene can also target the immunogens to all three types ofAPCs. The immune responses could be further boosted with use of the sameprotein antigens generated in vitro (i.e., “prime and boost”), ifnecessary. Using this strategy, specific cellular and humoral immuneresponses against infection of flavivirus were accomplished throughintramuscular (i.m.) injection of a DNA vaccine. (See Casares et al.,Viral. Immunol. 10:129-36 (1997)).

Vaccine Compositions

Therapeutic or prophylactic compositions are provided herein, whichgenerally comprise mixtures of one or more monoclonal antibodies orScFvs and combinations thereof. The prophylactic vaccines can be used toprevent a flavivirus infection and the therapeutic vaccines can be usedto treat individuals following a flavivirus infection. Prophylactic usesinclude the provision of increased antibody titer to a flavivirus in avaccination subject. In this manner, subjects at high risk ofcontracting flavivirus (i.e., in subtropical regions whereviral-carrying mosquitos thrive) can be provided with passive immunityto a flavivirus.

These vaccine compositions can be administered in conjunction withancillary immunoregulatory agents. For example, cytokines, lymphokines,and chemokines, including, but not limited to, IL-2, modified IL-2(Cys125→Ser125), GM-CSF, IL-12, γ-interferon, IP-10, MIP1β, and RANTES.

Methods of Immunization

The vaccines of the present invention have superior immunoprotective andimmunotherapeutic properties over other anti-viral vaccines.

The invention provides a method of immunization, e.g., inducing animmune response, of a subject. A subject is immunized by administrationto the subject a composition containing a membrane fusion protein of apathogenic enveloped virus. The fusion protein is coated or embedded ina biologically compatible matrix.

The fusion protein is glycosylated, e.g. contains acarbohydrate moiety.The carbohydrate moiety may be in the form of a monosaccharide,disaccharide(s). oligosaccharide(s), polysaccharide(s), or theirderivatives (e.g. sulfo- or phospho-substituted). The carbohydrate islinear or branched. The carbohydrate moiety is N-linked or O-linked to apolypeptide. N-linked glycosylation is to the amide nitrogen ofasparagine side chains and O-linked glycosylation is to the hydroxyoxygen of serine and threonine side chains.

The carbohydrate moiety is endogenous to the subject being vaccinated.Alternatively, the carbohydrate moiety is exogenous to the subject beingvaccinated. The carbohydrate moiety is a carbohydrate moieties that arenot typically expressed on polypeptides of the subject being vaccinated.For example, the carbohydrate moieties are plant-specific carbohydrates.Plant specific carbohydrate moieties include for example N-linked glycanhaving a core bound α1,3 fucose or a core bound β1,2 xylose.Alternatively, the carbohydrate moiety are carbohydrate moieties thatare expressed on polypeptides or lipids of the subject being vaccinate.For example many host cells have been genetically engineered to producehuman proteins with human-like sugar attachments.

For example, the fusion protein is a trimeric hemagglutinin protein.Optionally, the hemagglutinin protein is produced in a non-mammaliancell such as a plant cell.

The subject is at risk of developing or suffering from a viralinfection. Flavivirus family members include, for example West Nilevirus, Dengue virus (serotypes 1-4), St. Louis encephalitis virus,yellow fever virus, Japanese encephalitis virus, or Murray Valleyencephalitis virus. For example, the subject has traveled to regions orcountries in which other flaviviral infections have been reported.

The methods described herein lead to a reduction in the severity or thealleviation of one or more symptoms of a viral infection. Infections arediagnosed and or monitored, typically by a physician using standardmethodologies. A subject requiring immunization is identified by methodsknow in the art. For example subjects are immunized as outlined in theCDC's General Recommendation on Immunization (51(RR02) pp 1-36) Canceris diagnosed for example by physical exam, biopsy, blood test, or x-ray.

The subject is e.g., any mammal, e.g., a human, a primate, mouse, rat,dog, cat, cow, horse, pig, a fish or a bird.

The treatment is administered prior to diagnosis of the infection.Alternatively, treatment is administered after diagnosis.Efficaciousness of treatment is determined in association with any knownmethod for diagnosing or treating the particular disorder or infection.Alleviation of one or more symptoms of the disorder indicates that thecompound confers a clinical benefit.

Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a flavivirus-relateddisease or disorder. Such diseases or disorders include but are notlimited to, e.g., fever, meningitis, encephalitis, yellow fever, denguefever.

Prophylactic Methods

In one aspect, the invention provides methods for preventing aflavivirus-related disease or disorder in a subject by administering tothe subject a monoclonal antibody of the invention or an agentidentified according to the methods of the invention. For example,monoclonal antibody mAb-11, mAb11-LALA, and any variants thereof,wherein the Fc region is modified thereby reducing or abrogating bindingto the Fc-gamma receptor, may be administered in therapeuticallyeffective amounts. Optionally, two or more anti-flaviviruses antibodiesare co-administered.

Subjects at risk for a flavivirus-related diseases or disorders includepatients who have been exposed to the flavivirus from an infectedarthropod (i.e., mosquito or tick). For example, the subjects havetraveled to regions or countries of the world in which other flavivirusinfections have been reported and confirmed. Administration of aprophylactic agent can occur prior to the manifestation of symptomscharacteristic of the flaviviru-related disease or disorder, such that adisease or disorder is prevented or, alternatively, delayed in itsprogression.

The appropriate agent can be determined based on screening assaysdescribed herein. Alternatively, or in addition, the agent to beadministered is a monoclonal antibody that neutralizes a flavivirus thathas been identified according to the methods of the invention. In someembodiments, the antibody of the present invention can be administeredwith other antibodies or antibody fragments known to neutralizeflaviviruses. Administration of said antibodies can be sequential,concurrent, or alternating.

Therapeutic Methods

Another aspect of the invention pertains to methods of treating aflavivirus-related disease or disorder in a patient. In one embodiment,the method involves administering an agent (e.g., an agent identified bya screening assay described herein and/or monoclonal antibody identifiedaccording to the methods of the invention), or combination of agentsthat neutralize the flavivirus to a patient suffering from the diseaseor disorder.

Combinatory Methods

The invention provides treating a flavivirus-related disease ordisorder, such as West Nile fever, meningitis, Dengue fever, yellowfever or encephalitis, in a patient by administering two or moreantibodies, such as mAb11-LALA or a variant of mAb11, wherein the Fcregion of said variant does not bind or has reduced binding to the Fcgamma receptor, with other flavivirus neutralizing antibodies known inthe art, such as mAb11. In another embodiment, the invention providesmethods for treating a flavivirus-related disease or disorder in apatient by administering an antibody of the present invention, such asmAb11-LALA or a mAb11 variant as described herein, with any anti-viralagent known in the art. Anti-viral agents can be peptides, nucleicacids, small molecules, inhibitors, or RNAi.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: Prophylaxis Against Lethal Dengue-2 Virus Infectionin Mice

Here is described a study examining the prophylactic effect ofadministration of the mAb11 antibody (both wild-type and the mutatedform with modified Fc region) in mice. Forty mice were assessed in thisstudy, and were divided into 5 groups containing 8 mice each. In Group1, the mice were administered an isotype control IgG control antibody.Group 2 was administered 250 ug/mouse (˜12.5 mg/kg) of wild-type mAb11.Group 3 was administered 50 ug/mouse (˜2.5 mg/kg) of wild-type mAb11 (wtmAb). Group 4 was administered 250 ug/mouse (˜12.5 mg/kg) of mAb11 witha LALA mutation in its Fc region (mut mAb). Group 5 was administered 50ug/mouse (˜2.5 mg/kg) of mAb11 with a LALA mutation in its Fc region(mut mAb).

After a period of time after administration of the antibody treatment(control, wildtype mAb11 or mutant mAb11), for example, 4 hours or 24hours, the mice were injected with a lethal dose of Dengue-2 virus. Themice were then observed daily over 10 days for a variety of factors,such as weight loss, morbidity, and clinical scores that includeappearance (coat and eye appearance), mobility, and attitude (and asillustrated in the table below).

TABLE 1 Clinical Score Score Initials Description Appearance, Mobility,and Attitude 1 H Healthy Smooth coat and bright eyes. Active, scurrying,and burrowing. Alert 2 SR Slightly Slightly ruffled coat (usually aroundruffled head/neck). Active, scurrying, and burrowing. Alert 3 R RuffledRuffled coat throughout body - “wet” appearance. Active, scurrying, andburrowing. Alert 4 S Sick Very ruffled coat. Slightly closed, inseteyes. Walking, but no scurrying. Mildly lethargic 5 VS Very Very ruffledcoat. Closed, inset eyes. sick Slow to no movement. Will return uprightif put on its side. Extremely lethargic 6 E Euthanize Very ruffled coat.Closed, inset eyes. 7 D Deceased Moribund. Requires immediateeuthanasia. No move- ment or uncontrolled spastic movements. Will notreturn upright if put on its side. Completely unaware or in noticeabledistressMice were euthanized if they scored at 5 or above and/or when theyreached 20% weight loss.

Study results were quantified and presented in FIG. 3. Specifically,FIG. 3A shows the Kaplan-Meier curves demonstrating the survival rate ofthe different groups of mice. The results show that control antibody andboth dosages of wild-type mAb11 were not effective at protecting themice from disease-related death/euthanization. In contrast, all of themice receiving either dosage of the mutant mAb11 of the presentinvention survived the lethal dose infection. These results demonstratethe mutant mAb11 has prophylactic efficacy for flaviviral infection,especially compared to the wild-type antibody.

When comparing percent weight loss from before infection (FIG. 3B),animals administered mutant mAb11 lost less weight than animalsadministered control antibody or wild-type mAb11 lost weight over thecourse of the study. The clinical score analysis combine otherqualitative observations regarding the appearance and attitude of thesubjects to score the degree of observed health of the animals. As shownin FIG. 3C, the health of the animals receiving control or wild-typemAb11 quickly declined (clinical score numbers increased to above 4),while the animals that received the mutant mAb11 remained healthy,alert, and mobile even after 10 days of infection.

Taken together, all of these results indicate that the mutant mAb11effectively prevented flaviviral infection-associated death, delayedprogression of the symptoms of the disease, and had an overallprophylactic effect on mice infected with Dengue virus.

Example 2: Comparison Between Antibodies Derived from Mammalian Cellsand Plant Cells

As described herein, the antibodies of the present invention can beproduced in transgenic plants. Other studies have shown that humanizedantibodies suitable for administration for treatment in humans have beensuccessfully produced in plants. Moreover, therapeutic antibodyproduction in plants is an inexpensive and efficient alternative toantibody production in mammalian cells, and moreover, lacks animalpathogenic contaminants. To examine the efficacy of the mAb11 antibodyof the present invention (containing the LALA mutation in the Fc region;mutAb) the antibodies produced from mammalian expression system and fromplant (tobacco plant) were compared in vivo.

A129 mice were used for in this study. A129 mice lack IFN at/3receptors, which are required for restricting viral replication in thecentral nervous system. A129 mice represent the most stringent model inthe field for recapitulating human disease with regard to flaviviralinfection. A129 mice infected with 1 PFU (plaque forming units) ofDengue virus causes paralysis. (Prestwood et al., J. Virol, 2012,

Forty-five mice were assessed in this study, and were divided into 5groups containing 9 mice each. In Group 1, the mice were administered acontrol IgG antibody. The control antibody used was Z-MAB (Zero-bindingmonoclonal antibody; AB Biosciences). Group 2 was administered 250ug/mouse (˜12.5 mg/kg) of mAb11 with a LALA mutation in its Fc region(mut mAb) produced in a mammalian expression system (mutAb mammalian).Group 3 was administered 50 ug/mouse (˜2.5 mg/kg) of mutant mAb11produced in a mammalian expression system. Group 4 was administered 250ug/mouse (˜12.5 mg/kg) of mutant mAb produced in a plant expressionsystem (mutAb plant). Group 5 was administered 50 ug/mouse (˜2.5 mg/kg)of mutant mAb11 produced in a plant expression system.

Mice were administered in the dosages of antibody as described above,then challenged with a lethal dose of Dengue virus. The mice were thenobserved daily over 20 days for a variety of factors, such as weightloss, morbidity, and clinical scores that include appearance (coat andeye appearance), mobility, and attitude (as described in Table 1).

The results of these studies are summarized in FIGS. 4, 5 and 6. FIG. 5shows the overall survival of mice. Both doses of mutant mAb11 producedfrom mammalian systems demonstrated protective effect against viralinfection, compared to control (FIG. 4A). Similar to the results shownin Example 1 and FIG. 3, all mice administered the mutAb antibodysurvived beyond day 10 of the study. Both doses of mutAb from plant alsodemonstrated protective effect, as shown in Figure B. Comparison betweenthe two mutAb from mammalian and plant is shown in FIG. 3, which showsthat the mutAb from plants were just as effective, if not more so, inprotecting the mice from disease progression and death.

FIG. 5 shows the percent weight loss in animals over the course of thestudy. Both mutAb mammalian and mutAb plant antibodies both protectedmice from weight loss in comparison to the control antibody (FIGS. 5Band 5C). Comparison between mammalian and plant-derived mutAb showed nosignificant difference between weight loss as a result of the antibodyproduction method (FIG. 5A).

FIG. 6 shows the clinical scores of the animals over the course of thestudy. Administration of mutAb mammalian and mutAb plant antibodiesprotected mice from progression or severity of symptoms compared tocontrol antibody (FIGS. 6B and 6C). Comparison between mammalian andplant-derived mutAb showed that the two antibodies performed similarly,with the mice receiving the higher dosage of plant-derived mutAb (250ug) showing slightly better overall health as measured by the clinicalscores (FIG. 6A).

Taken together, these results show that production of the mutantantibodies of the present invention were just as effective at protectionand reducing severity of the disease as the antibodies produced bystandard mammalian expression systems. For some of the measuredparameters, particularly at high doses of the mutAb plant-derivedantibody, the plant-derived antibodies showed to have a slightlyincreased therapeutic effect in comparison to the mammalian-derivedantibodies. Thus, plant-derived antibodies of the present inventionwould be useful for the protection and treatment of flaviviralinfection.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. An isolated humanized monoclonal antibodycomprising: a V_(H) amino acid sequence having SEQ ID NO: 1, a V_(L)amino acid sequence having SEQ ID NO: 3, and a modified Fc region havingmutations at amino acid positions 234 and 235, such that the Fc regiondoes not bind to the Fcγ receptor, wherein the mutations are L234A andL235A wherein positions 234 and 235 corresponds to positions 240 and 241of SEQ ID NO: 20; and wherein the antibody binds and neutralizes aflavivirus.
 2. An isolated humanized monoclonal antibody comprising: aV_(H) encoded by the nucleotide sequence SEQ ID NO: 2, a V_(L) encodedby the nucleotide sequence SEQ ID NO: 4, and a modified Fc region havingmutations at amino acid positions 234 and 235, such that the Fc regiondoes not bind to the Fcγ receptor, wherein the mutations are L234A andL235A wherein positions 234 and 235 corresponds to positions 240 and 241of SEQ ID NO: 20; and wherein the antibody binds and neutralizes aflavivirus.
 3. The antibody of claim 1 or claim 2, wherein said antibodydoes not contribute to an antibody-dependent enhancement of a flavivirusinfection.
 4. The antibody of claim 1 or 2, wherein the Fc regioncomprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO:
 13. 5.The antibody of claim 1 or claim 2, wherein the modified Fc region bindsto the neonatal Fc receptor.
 6. The antibody of claim 1 or claim 2,wherein the flavivirus is West Nile virus, Dengue virus (serotypes 1-4),St. Louis encephalitis virus, yellow fever virus, Japanese encephalitisvirus, and Murray Valley encephalitis virus.
 7. The antibody of claim 1or claim 2, wherein said humanized monoclonal antibody is produced in aplant.
 8. The antibody of claim 1 or claim 2 linked to a therapeuticagent.
 9. The antibody of claim 8, wherein said therapeutic agent is atoxin, a radiolabel, a siRNA, a small molecule, or a cytokine.
 10. Theantibody of claim 9, wherein said cytokine is TGF-beta.
 11. An isolatedcell producing the antibody of claim 1 or claim
 2. 12. A method ofreducing antibody-dependent enhancement of a flavivirus infectioncomprising administering the antibody of claim 1 or claim 2 to asubject.
 13. The method of claim 12, wherein said antibody isadministered after a first infection by a flavivirus.
 14. A method oftreating or alleviating a symptom of a flavivirus infection, comprisingadministering to a subject in need thereof a composition comprising anantibody according to claim
 1. 15. A method of delaying the onset of oneor more symptoms of a flavivirus infection, comprising administering toa subject in need thereof a composition comprising an antibody accordingto claim
 1. 16. The method of any claim 14, wherein said one or moresymptom comprises weight loss, paralysis, fever, headache, nausea,vomiting, skin rash, and body aches.
 17. The method of claim 12, whereinsaid flavivirus is West Nile virus, Dengue virus (serotypes 1-4), St.Louis encephalitis virus, yellow fever virus, Japanese encephalitisvirus, or Murray Valley encephalitis virus.
 18. A nucleic acid sequencecomprising a nucleic acid sequence of SEQ ID NO: 2, 4 and
 8. 19. Anucleic acid sequence encoding a polypeptide comprising SEQ ID NO: 1, 3and
 7. 20. A polypeptide comprising the amino acid sequence comprisingSEQ ID NO: 1, 3 and
 7. 21. A vector comprising the nucleic acid of claim18 or
 19. 22. An isolated cell comprising the vector of claim
 21. 23.The isolated cell of claim 11, wherein said isolated cell is a plantcell.
 24. The method of claim 15, wherein said one or more symptomcomprises weight loss, paralysis, fever, headache, nausea, vomiting,skin rash, or body aches.
 25. The method of claim 14, wherein saidflavivirus is West Nile virus, Dengue virus (serotypes 1-4), St. Louisencephalitis virus, yellow fever virus, Japanese encephalitis virus, orMurray Valley encephalitis virus.
 26. The method of claim 15, whereinsaid flavivirus is West Nile virus, Dengue virus (serotypes 1-4), St.Louis encephalitis virus, yellow fever virus, Japanese encephalitisvirus, or Murray Valley encephalitis virus.
 27. A nucleic acid sequenceencoding a polypeptide comprising SEQ ID NOS: 1, 3 and
 13. 28. Apolypeptide comprising the amino acid sequence comprising SEQ ID NOS: 1,3, and
 13. 29. A method of treating or alleviating a symptom of aflavivirus infection, comprising administering to a subject in needthereof a composition comprising an antibody according to claim
 2. 30. Amethod of delaying the onset of one or more symptoms of a flavivirusinfection, comprising administering to a subject in need thereof acomposition comprising an antibody according to claim
 2. 31. Theisolated cell of claim 22, wherein said isolated cell is a plant cell.32. The method of claim 16, 17, 29, or 30 further comprisingadministering an anti-viral agent.
 33. The method of claim 32, whereinthe anti-viral agent is an antibody, an antibody linked to a therapeuticagent, or a small molecule.
 34. The method of claim 32, wherein saidantibody and the anti-viral agent are administered sequentially orconcurrently.